DESCRIPTION: Most mitochondrial proteins are encoded in the nucleus, synthesized in the cytoplasm, and, subsequently, imported into the mitochondria. During this import process, proteins are sorted to the matrix, inner membrane, intermembrane space, or outer membrane. Although most to the components facilitating the import of proteins into the mitochondrial matrix have now been identified, the proteins and molecular mechanisms that mediate sorting and integration of precursors into the mitochondrial membranes are poorly understood. The goal of the research described by this proposal is to identify and characterize such intramitochondrial sorting components. The first specific aim is to identify yeast mutants that restore correct sorting to a protein containing a defective inner membrane sorting signal. The genes defined by such mutations will be identified and their analysis should lead to the characterization of proteins that interact with sorting signals. A second, parallel specific aim is to identify sorting components through the analysis proteins that are crosslinked to a precursor which is arrested during the import process. The third specific aim is to compare the mechanisms of protein sorting to outer and inner membranes. This goal will involve the analysis of import of a number of fusion constructs that contain portions of both inner and outer membrane proteins and will assess the role in sorting of position of the sorting signal, membrane potential, and nature of the targeting presequence.